Growth cones are the motile tips of growing axons. During development they guide axons to their targets by extension, retraction, pausing and turning behaviors in response to environmental cues. Axon branching, resulting from growth cone pausing, is an important form of axon guidance. Ultimately, dynamic reorganization of the actin and microtubule cytoskeleton underlies behaviors of the growth cone required for axon guidance and branching. However, the intracellular signaling pathways by which guidance cues regulate cytoskeletal dynamics in the growth cone are not well understood. The long-term goal of this work is to elucidate mechanisms of axon guidance in the mammalian central nervous system (CNS). This work will be carried out on developing cortical neurons and is highly relevant to the health-related goal of promoting axon sprouting and regeneration after CNS injury. The first specific aim is to analyze with live cell imaging how guidance molecules influence the organization of the cytoskeleton to bring about growth cone behaviors. To follow dynamic cytoskeletal changes over time, actin filaments and microtubules will be labeled by microinjection of fluorescently-tagged phalloidin and tubulin into dissociated cortical neurons from newborn rodents. Localized application of the guidance molecules Semaphorin 3A, Slits 1, 2, 2-N and FGF-2 to the growth cone will be used to elicit localized cytoskeletal changes leading to changes in growth cone behaviors and axon branching. The second specific aim is to investigate the functions of the Mena/VASP proteins, a family of actin regulatory proteins thought to play a role in growth cone pausing and branching at choice points in the CNS. Dynamic changes in the association of Mena/VASP proteins with different populations of actin filaments in cortical growth cones will be analyzed in time-lapse microscopy during growth cone behaviors elicited by different guidance cues. The Mena/VASP proteins will be labeled by infecting cortical neurons with viral constructs of the proteins tagged with green fluorescent protein (GFP). The third specific aim is to address the mechanisms by which intracellular calcium signaling regulates growth cone behaviors. In dissociated cortical neurons calcium indicator dyes will be used to study the role of calcium transients in cytoskeletal reorganization. Calcium imaging of living slices of the developing cortex will be used to determine whether calcium transients in vivo regulate axon outgrowth. Taken together this work will advance our understanding of how the developing brain forms appropriate connections [unreadable] [unreadable]